Apoptosis inhibitors for treating neurodegenerative diseases

ABSTRACT

Apoptotic cell death in a fully differentiated, non-dividing cell such as a neuron is caused by an abortive attempt of the cell to re-enter or pass through the mitotic cycle. Therefore, agents which prevent such entry or passage are effective in preventing, or at least delaying, apoptotic cell death and are therefore useful in the treatment of neurodegenerative diseases in general, including stroke, Alzheimer&#39;s disease, Parkinson&#39;s disease and motor-neuron disease in particular.

This application is a continuation of Ser. No. 08/556,974, filed May 8,1996, now U.S. Pat. No. 5,840,719, which is a 371 of PCT/GB94/01169,filed May 31, 1994.

This invention relates to the prevention or at least delay of death ofcells, particularly non-dividing cells.

Controlling cell death is useful in the treatment of neurodegenerativediseases in general, including stroke, Alzheimer's disease, Parkinson'sdisease and motor-neuron disease in particular.

Neurons are examples of cells which have terminally differentiated andtherefore are non-dividing. There are two general types of neuronal celldeath: necrotic and apoptotic. The two differ in terms of initiatingfactors, morphological changes, time course and mechanism. Necrotic celldeath is often seen in situations in which there is excess calcium ioninflux caused, for example, by application of the excitatoryneurotransmitter glutamate. Apoptotic cell death is sometimes referredto as programmed cell death and is seen as a normal developmental eventin the nervous system and in other tissues. Fully differentiated neuronsare normally dependent on a trophic factor for their survival andapoptotic death can be triggered by trophic factor withdrawal.

The role of apoptotic death in neurodegenerative disease has not beenfully established. However, there are a variety of diseases of thenervous system in which neurons destined to die can be rescued byapplication of appropriate growth factors. While the cause of a cell'sfate may vary from disease to disease, it seems likely that the finaldeath pathway is apoptosis in each case.

The observation that the application of appropriate growth factors canrescue neurons destined to die has given rise to proposals for treatmentfor certain neurodegenerative diseases in the past. However, because thetreatment is based on the use of peptide growth factors, which areunable to cross the blood-brain barrier, most current clinical trialsfor neurodegenerative disease are for diseases of the peripheral, ratherthan central, nervous system. Furthermore, such individual treatments ashave been proposed are rather disease-specific; there has not hithertoexisted the basis for developing a more generally applicable therapeuticor prophylactic regime for the management of neurodegenerative diseaseand other diseases involving the cell death of non-dividing cells.

It has now been discovered that cell death in a fully differentiated,non-dividing cell such as a neuron appears to be the result of anabortive attempt of the cell to re-enter or pass through the mitoticcycle. Therefore, agents which prevent entry into or passage through themitotic cycle should be effective in preventing, or at least delaying,apoptotic cell death.

According to a first aspect of the present invention, there is provideda method of treating or preventing a disease involving apoptotic celldeath, the method comprising administering to a subject, or to cells ofa subject, an effective amount of an agent which prevents cell entryinto or passage through the mitotic cycle; provided that the agent isnot bFGF, IGF-I, IGF-II, potassium ions or a cAMP-elevating agent.

The mitotic cell cycle has four distinct phases, G₁, S, G₂ and M. Thebeginning event in the cell cycle, called start, takes place in the G₁phase and has a unique function. The decision or commitment to undergoanother cell cycle is made at start. Once a cell has passed throughstart, it passes through the remainder of the G₁ phase, which is thepre-DNA synthesis phase. The second stage, the S phase, is when DNAsynthesis takes place. This is followed by the G₂ phase, which is theperiod between DNA synthesis and mitosis. Mitosis itself occurs at the Mphase. Fully differentiated cells such as neurons are generally regardedas not being in any of these four phases of the cycle; they are usuallydescribed as being in a G₀ state, so as to indicate that they would notnormally progress through the cycle.

Preferred agents useful in the invention may totally prevent entry intothe cycle. Other agents may allow passage through the cycle to a certainextend but not completely. In some cases it well be preferred to blockpassage through the cycle relatively early on; in other cases, blockinglater may be more appropriate. Generally, agents may block the cycle atG₁, G₁/S, S or S/G₂.

Agents which may totally prevent entry into the cycle may includeFK-506.

Agents which block the cell cycle at the G₁ phase include agents whichinterfere with early gene expression or which interfere with the properfunctioning of the products of early expressed genes. Examples of earlyexpressed genes include c-fos, c-jun and c-myc. Agents which prevent thephosphorylation state of the retinoblastoma (Rb) protein may also blockthe cycle at this early stage.

The cell cycle may be stopped at the late G₁ stage by agents whichinterfere with the expression or proper functioning of cyclin D or E.(Interference, in this and other cases, may be direct or indirect).Correspondingly, the cell cycle may be stopped at the G₁/S stage byagents which interfere with the expression or proper functioning ofcyclin A and/or D. Other agents which block the cell cycle at G₁/Sinclude those which interfere (directly or indirectly) with theactivation of the enzymes cdc2, cdk2 and/or cdk4; the activated form ofcdk2 has been shown to be necessary for normal passage through the Sphase.

The principal way of preventing passage through the S phase is of courseto block DNA synthesis. Many agents useful for this purpose, includingthe use of nucleotide analogues such as dideoxynucleotides and the useof inhibitors of DNA polymerase, are known in the art.

Among agents useful in blocking the cell cycle at the G₂ phase are thosewhich inhibit, directly or indirectly, inactivation of the enzymes cdc2and cdk2.

The cell cycle may be stopped at G₂/M stage by agents which interferewith the expression or proper functioning of cyclin G and/or those whichinhibit the reactivation of the enzyme cdc2.

Finally, the cycle may be stopped at the M phase by interfering with theproper processing and organisation of tubulin.

Generally speaking, it is possible to monitor the stage in the mitoticcycle at which the passage of cells have been stopped by looking for theexpression, proper functioning, activation or inhibition, as the casemay be, of the various genes and enzymes discussed above. In addition,in the S phase, the extent of DNA synthesis taking place can be assessedby any suitable method such as might be used for assessing DNA synthesisin other situations. Examples include measuring the incorporation of adetectable nucleotide, such as bromodeoxyuridine (BrdU), andfluorescence-activated cell sorting (FACS) analysis based on theintercalation of a suitable fluorescent dye, such as propidium iodide.In fact, not only does the extent of DNA synthesis serve as a marker ofwhether cells are in the S or subsequent phases of the mitotic cellcycle (in which case the cells have a double DNA complement), it alsoserves as a marker of whether cells are undergoing cell death (in whichcase the DNA complement of the cells is less than the normal, singlelevel). An additional marker of cells being in the M phase is providedby the gross morphological changes (including chromosome condensationand nuclear envelope breakdown) which are taking place then.

To summarise, there are a number of different ways in which agentsuseful in the invention may prevent entry into the mitotic cell cycle.First, DNA synthesis could be inhibited. Secondly, cell divisioncycle-associated enzymes could be inhibited. Many of these enzymes areencoded by the cdc series of genes; additionally, many of the enzymesinvolved are kinases or phosphatases. Either the enzyme itself may beinhibited, or its activation, from a precursor, could be inhibited.Equally, entry into the mitotic cycle can be measured in a number ofdifferent ways. Markers of DNA synthesis can be used; or other cellcycle markers can be examined. Such cell cycle markers include cyclinsynthesis, cdc2 or cdk2 activation, early gene expression andredistribution or modification of certain transcription factors.

Broadly speaking, compounds can be assessed for their potentialusefulness in the invention by their ability to inhibit in vitro, as amodel for their activity in vivo, any of the processes of the cell cycledescribed above. Enzyme inhibition assays, based on the enzymes involvedin the cycle, can therefore act as screens for useful compounds.

One particular way of assessing candidate compounds for their usefulnessof the invention is to use the following model. PC12 ratpheochromocytoma cells are adrenal chromaffin-like cells which have theability to differentiate terminally into a sympathetic neuron-likephenotype in response to nerve growth factor (NGF). If thesedifferentiated cells are subsequently withdrawn from NGF, they undergo aseries of morphological and biochemical changes that are characteristicof cell death by apoptosis. Compounds can be screened by assessing theirability to prevent PC12 cell death following NGF withdrawal. FIG. 1 ofthe accompanying drawings shows the life and death of a PC12 cell. Inthe presence of serum alone, these cells will divide and survive. In thepresence of NGF or NGF plus serum, they will differentiate and survive.But in the absence of NGF and serum, the cells die unless a compounduseful in the invention is present).

In principle, any agent which prevents cell entry into or passagethrough the mitotic cycle could be useful in the invention. Practicalconsiderations dictate that compounds useful in the invention will havea sufficiently low toxicity to result in a useful therapeutic index.Preferred compounds useful in the invention will be those which arereadily formulatable and pharmaceutically (or veterinarily) acceptableformulations; however, continuing advances in formulation technology arelikely to result in a few, if any, real limitations in this respect.Preferred compounds useful in the invention as able to cross theblood-brain barrier, as they would then be useful in preventing celldeath in neurons of the central nervous system. Even if compounds cannotcross the blood-brain barrier, though, they may still be useful in theinvention in preventing cell death in neurons of the peripheral nervoussystem and other, non-neuronal, cell types.

One particular cell-associated kinase whose activation may usefully beinhibited in the practice of the present invention is p34^(cdc2).Inhibitors of the activation of p34^(cdc2) kinase include6-dimethylaminopurine (6-DMAP); analogues of 6-DMAP and other compoundsmay also be used in the invention.

FIG. 2 of the accompanying drawings shows a hypothetical frameworkshowing the complexity of potential interactions among various cellcycle elements during neuronal apoptosis initiated by NGF withdrawal ina model system. This scheme is based on a situation in which cdc2 kinaseis activated in the absence of NGF and leads to neuronal apoptosis,perhaps by modification of structural elements such as the nuclearenvelope. Partly as a result of this complexity, a large number ofapproaches to selecting compounds useful in the invention becomesapparent. cdc2 is normally phosphorylated and inactivated by wee1 andmik1 kinases and activated by cdc25 phasphatase. It also requires anassociated regulatory subunit, such as the G₂/M cyclin B for activity.c-mos is included as a potential regulatory factor that interacts withcyclic AMP via protein kinase A (PKA) and Ca²⁺, known to be neuronalsurvival agents. So, just considering classes of compounds useful in theinvention as a result of their interference with the normal functioningof cdc2, it can be seen that the following, among others, are useful:

promoters or activators of wee1 and/or mik1;

inhibitors of cdc25; and/or

antagonists of cyclin B.

Among the specific compounds that have been shown to be capable ofpreventing cell death are serotonin (>10 mM), dopamine (>10 mM),ascorbin acid (>100 mM), gluquidone (>10 mM), caffeine (>1 mM) and highdoses of the steroids hydrocortisone (0.1 to 100 μM) and dexamethasone(>0.1 mM).

The use of bFGF, insulin-like growth factors I and II (IGF-I andIGF-II), depolarisation with potassium ions and certain cAMP elevatingagents including certain cAMP-analogues is excluded from the inventionas defined herein because these specific agents have previously beenshown to prevent neuronal cell death. For example: bFGF has been shownto support the survival of cerebral cortical neurons in primary cultureby Morrison et al. (Proc. Nat'l. Acad. Sci. USA 83 7537-7541 (1986));IGF-I and IGF-II have been shown to rescue PC12 cells from serum-freedeath by Rukenstein et al. (J. Neurosci. 11 2552-2563 (1991)); high K⁺concentrations have been shown to support the survival of chick embryosympathetic neurons by Wadake et al. (Exp. Cell. Res. 144 377-384(1983)); and the following cAMP elevating agents have been shown toprevent cell death:

8-(4-chlorophenylthio)-adenosine-3′:5′-cyclic-menophosphate (CPT-cAMP)(see, for example, Koike Prog. Neuro-Psychopharmacol. and Biol.Psychiat. 16 95-106 (1992)),

CPT-cAMP, forskolin, isobutyl methylxanthine (IBMX) and cholera toxin(see Martin et al. J. Neurobiol. 23 1205-1220 (1992)) and

8-bromo-cAMP, N⁶,O^(2′)-dibutyryl-cAMP and N⁶,O^(2′)-dioctanoyl-cAMP(see Rydel and Greene Proc. Nat'l. Acad. Sci. USA 85 1257-1261 (1988)).

However, it was not until the present invention was made that the modeof action of the above compounds, as an apparently unrelated group ofstructurally and functionally diverse compounds, could satisfactorily beexplained by a unified theory. There was therefore no motivation, beforethis invention, to seek other agents which also prevent cell entry into,or successful passage through, the mitotic cycle.

The invention is in general useful in preventing cell death in allnon-dividing cells. Examples include muscle cells (such as striatedmuscle cells and heart myocytes (Nazareth et al. J. Mol. Cell. Cardiol.23 1351-1354 (1991)) and neurons.

While the invention has particular application in the treatment andprophylaxis of disease in humans, there is no reason in principle why itshould not be applied to animals of other species.

Compounds useful in the invention may, depending on their physical andchemical properties, be administered on their own but will generally beformulated with a pharmaceutically or veterinarily acceptable carrier.The preferred mode of long term treatment of degenerative disease,particularly neurodegenerative disease, is to use agents that arebioactive and bioavailable when administered enterally (particularlyorally), so orally administrable formulations are of particularimportance. If a neurodegenerative disease of the central nervous systemis to be treated by means of an oral formulation, the agent shouldadditionally be able to cross the blood-brain barrier. Agents whichcannot cross the blood-brain barrier, such as certain peptides andpolypeptides, are not necessarily precluded from being useful in theinvention, even in the treatment of neurodegenerative disease, as theymay be administered locally, for example by the use of an Omayareservoir or, in the case of peptides and polypeptides, by theimplantation of appropriate cells expressing and secreting the factor.

The dosage of any particular agent will depend on a number of factorsand is likely to be optimised in experimental or clinical trials. In anyevent, the appropriate dosage for a particular patient or subject willbe determined by the responsible physician or clinician in each case.

The invention extends to the use of an agent (other than bFGF, IGF-I,IGF-II, potassium ions or a cAMP-elevating agent) which prevents cellentry into, or passage through, the mitotic cycle in the preparation ofa medicament for treating or preventing a disease involving apoptoticcell death.

Diseases treatable by means of the invention include neurodegenerativediseases in general, including stroke, Alzheimer's disease, Parkinson'sdisease and motor-neuron disease in particular.

The invention may also have application in non-medical fields. Accordingto a second aspect of the invention, there is provided a method ofpreventing apoptotic cell death, the method comprising administering tocells an effective amount of an agent which prevents cell entry into themitotic cycle, provided that the agent is not bFGF, IGF-I or IGF-II,potassium ions or a cAMP-elevating agent.

Such a method may be useful in laboratory modelling studies, possibly inresearch on the aetiology of cell death.

Preferred features of the second aspect of the invention are as for thefirst aspect, mutatis mutandis.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be illustrated by the following examples. Theexamples refer to the drawings, in which:

FIG. 1 shows schematically the life and death of a PC12 cell;

FIG. 2 shows a hypothetical framework showing the complexity ofpotential interactions among various cell cycle elements during neuronalapoptosis initiated by NGF withdrawal.

FIG. 3 relates to Example 2 and is an autoradiograph of a 12.5%SDS-polyacrylamide gel showing that p34^(cdc2) kinase is activated inconditions leading to apoptosis;

FIG. 4 relates to Example 3 and is a bar chart showing that serotoninpromotes cell survival in conditions leading to apoptosis;

FIG. 5 relates to Example 4 and is a bar chart showing that dopaminepromotes cell survival in conditions leading to apoptosis;

FIG. 6 relates to Example 5 and is a bar chart showing that L-ascorbicacid promotes cell survival in conditions leading to apoptosis;

FIG. 7 relates to Example 6 and is a bar chart showing that gliquidonepromotes cell survival in conditions leading to apoptosis;

FIG. 8 relates to Example 7 and is a bar chart showing that caffeinepromotes cell survival in conditions leading to apoptosis;

FIG. 9 relates to Example 8 and is a bar chart showing thathydrocortisone promotes cell survival in conditions leading toapoptosis;

FIG. 10 relates to Example 9 and is a bar chart showing thatdexamethasone promotes cell survival in conditions leading to apoptosis;

FIG. 11 relates to Example 10 and is a bar chart showing that5-dimethylaminopurine promotes cell survival in conditions leading toapoptosis;

FIG. 12 relates to Example 11 and is a bar chart showing thatdipyridamole promotes cell survival in conditions leading to apoptosis;

FIG. 13 relates to Example 12 and is a bar chart showing that milrinonepromotes cell survival in conditions leading to apoptosis;

FIG. 14 relates to Example 13 and is a bar chart showing that minoxidilsulphate promotes cell survival in conditions leading to apoptosis.

FIG. 15 relates to the Additional Experiment and shows thatstaurosporine induces a dose-dependent decrease in survival ofdifferentiated PC12 cells in the presence of NGF;

FIG. 16 relates to the Additional Experiment and shows the effect ofstaurosporine on cdc2 activity and cell death in PC12 cells;

FIG. 17 relates to the Additional Experiment and shows the effect ofoverexpression of bcl-2 on staurosporine-induced cdc2 kinase activity inproliferating human fibroblasts;

FIGS. 18A and 18B relates to the Additional Experiment and shows theeffect of bcl-2 overexpression on activation of cdc2 induced bystaurosporine in quiescent rat1 fibroblasts; and

FIG. 19 relates to the Additional Experiment and shows the effect of thebcl-2 overexpression on staurosporine-induced cdc2 kinase activity inquiescent Swiss 3T3 fibroblasts.

PREPARATION

Maintenance and Differentiation of PC12 Cells

PC12 rat pheochromocytoma cells (Greene and Tischler Proc. Nat'l. Acad.Sci. USA 73 2424-2428 (1976)), obtained from Dr. P. Doherty, Departmentof Experimental Pathology, United Medical and Dental School, Guy'sHospital, London Bridge, London SE1 9RT, where maintained in anundifferentiated state by seeding at a density of 8×10⁴ cells/cm² onto acollagen substrate in SATO medium containing 2% heat-inactivated foetalcalf serum (GIBCO) and 10 mg/l insulin (Sigma) in a humidifiedatmosphere at 37° C. in 8% CO₂. Cells were re-fed every 3-4 days. (SATOmedium is Dulbecco's Modified Eagles' Medium (DMEM) containing:

4.32 g/l BSA (Pathocyte 4; ICN),

0.075 mg/l progesterone (Sigma),

20.0 mg/l putrescine (Sigma),

0.5 mg/l L-thyroxine (Sigma),

0.05 mg/l selenium (Sigma),

0.4175 mg/l tri-iodo-thyronine (Sigma),

125 mg/l transferrin (Sigma) and

1.25 mM glutamine (GIBCO).)

PC12 cells were differentiated by passaging into serum- and insulin-freeSATO medium containing 100 ng/ml nerve growth factor (NGF) (purifiedfrom male submaxillary glands as described (Winter et al. Neuron 1973-981 (1988) and Suda et al. Proc. Nat'l. Acad. Sci. USA 75 4042-4046(1978)). Cells were plated onto polylysine-(16 μg/ml) andcollagen-coated dishes at a density of 8×10⁴ cells/cm² and weremaintained in a humidified atmosphere at 37° C., 8% CO₂, for 7 days.Cells were re-fed every 3 days.

EXAMPLE 1

Cells Undergoing Apoptosis Activate p34^(cdc2), a Kinase Specific to theCell Division Cycle

PC12 cells were plated onto polylysine/collagen-coated 90 mm plates at adensity of 2×10⁶ cells/dish, and were allowed to differentiate for 7days as in the Preparation.

After washing twice with DMEM cells were incubated in SATO mediumcontaining anti-NGF antibody for various periods of time. Medium wasremoved from the cells, and any floating cells were pelleted bycentrifugation at 1000 rpm for 10 min. Adherent cells were washed oncewith PBS at 4° C., and then lysed in 0.5 ml ice cold lysis buffer (50 mMTris/HCl pH 7.4, 0.25M NaCl, 0.1% NP-40, 5 mM EDTA, 50 mM NaF, 1 mMsodium orthovanadate, 1 mM sodium pyrophosphate, 50 μg/ml PMSF, 10 μg/mlTPCK, 10 μg/ml soy bean trypsin inhibitor, 1 Mg/ml aprotinin, 1 μg/mlleupeptin). An aliquot of the lysate was used to resuspend the cellspelleted by centrifugation, and was then combined with the lysate fromthe adherent cells. After 30 min incubation on ice, the suspension wascentrifuged at 13,000 g for 5 min at 4° C., and the supernatantcontaining cdc2 kinase was collected. Samples were adjusted to a finalvolume of 750 μl containing equal protein, and were then pre-clearedwith 40 μl protein A-SEPHAROSE beads (Boehringer Mannheim) by incubationon a rotating wheel at 4° C. for 1 h. The beads were then removed bycentrifugation for 15 s at 13,000 g, and the supernatant was thenincubated with 1:100 dilution of anti-cdc2 antibody for 2 h at 4° C.(The anti-cdc2 antibody was raised in rabbits to a peptide containingthe eight C-terminal amino acids of the human cdc2 protein(NH₂-CGGLDNQIKKM-COOH) and was a gift from Dr. C. Barth, Imperial CancerResearch Fund, Lincoln's Inn Fields, London WC2A 3PX.) Thecdc-2/antibody complex was then bound to protein A-SEPHAROSE beads byincubation for 40 μl beads for 1 h at 4° C. The complex was thenpelleted by centrifugation for 15 s at 13,000 g. After washing 4 timesin 1 ml lysis buffer, the pellet was washed once in PKA wash buffer (50mM Tris/HCl pH 7.4, 10 mM MgCl₂, 1 mM DTT), and then resuspended in 25μl of the same buffer containing 125 μg/ml histone H1. After 2 minincubation at 30° C., the reaction was initiated by the addition of 5 μlATP mix (1 μM ATP, 10 μCi/μl [γ-³²P]ATP in PKA wash buffer), and sampleswere incubated at 30° C. for 10 min. The reaction was terminated by theaddition of an equal volume of 2×Laemmli sample buffer and by incubatingfor 3 minutes at 100° C. Samples were separated on a 12.5% SDSpolyacrylamide gel. After drying, the gel was exposed to X-ray film, andkinase activity was assessed by the extend of incorporation of ³²P intohistone H1. The results are shown in FIG. 3. Phosphorylated histone H1can be visualised as a doublet migrating at ˜35 kDa some 16 hours afterNGF withdrawal.

EXAMPLE 2

Serotonin Promotes Cell Survival Under Conditions Leading to Apoptosis

PC12 cells were plated onto polylysine/collagen-coated 96 wellmicrotitre plates at a density of 7.5×10³/well and were allowed todifferentiate for 7 days in SATO medium containing 100 ng/ml NGF. Afterwashing one with DMEM, cells were incubated in SATO medium containing62.5 ng/ml anti-NGF antibody Boehringer Mannheim) and variousconcentrations of serotonin for various periods of time. Viability wasthen measured by the ability of cells to take up and metabolise theyellow dye MTT to the dark blue compound MTT-formazan. This product wasthen detected spectrophotometrically. The results are shown in FIG. 4.

EXAMPLE 3

Dopamine Promotes Cell Survival in Conditions leading to Apoptosis

The experiment of Example 2 was repeated, except that variousconcentrations of dopamine were used in place of serotonin. The resultsare shown in FIG. 5.

EXAMPLE 4

L-Ascorbin Acid Promotes Cell Survival in Conditions leading toApoptosis

The experiment of Example 2 was repeated, except that variousconcentrations of L-ascorbic acid were used in place of serotonin. Theresults are shown in FIG. 6.

EXAMPLE 5

Gliquidone Promotes Cell Survival in Conditions leading to Apoptosis

The experiment of Example 2 was repeated, except that variousconcentrations of gliquidone were used in place of serotonin. Theresults are shown in FIG. 7.

EXAMPLE 6

Caffeine Promotes Cell Survival in Conditions leading to Apoptosis

The experiment of Example 2 was repeated, except that variousconcentrations of caffeine were used in place of serotonin. The resultsare shown in FIG. 8.

EXAMPLE 7

Hydrocortisone Promotes Cell Survival in Conditions leading to Apoptosis

The experiment of Example 2 was repeated, except that variousconcentrations of hydrocortisone were used in place of serotonin. Theresults are shown in FIG. 9.

EXAMPLE 8

Dexamethasone Promotes Cell Survival in Conditions leading to Apoptosis

The experiment of Example 2 was repeated, except that variousconcentration of examethasone were used in place of serotonin. Theresults are shown in FIG. 10.

EXAMPLE 9

6-Dimethylaminopurine Promotes Cell Survival in Conditions leading toApoptosis

The experiment of Example 2 was repeated, except that variousconcentrations of 6-dimethylaminopurine (6-DMAP) were used in place ofserotonin. The results are shown in FIG. 11.

EXAMPLE 10

Dipyridamole Promotes Cell Survival in Conditions leading to Apoptosis

The experiment of Example 2 was repeated, except that variousconcentrations of dipyridamole were used in place of serotonin. Theresults are shown in FIG. 12.

EXAMPLE 11

Milrinone Promotes Cell Survival in Conditions leading to Apoptosis

The experiment of Example 2 was repeated, except that variousconcentrations of milrinone were used in place of serotonin. The resultsare shown in FIG. 13.

EXAMPLE 12

Minoxidil Sulphate Promotes Cell Survival in Conditions leading toApoptosis

The experiment of Example 2 was repeated, except that variousconcentrations of minoxidil sulphate were used in place of serotonin.The results are shown in FIG. 14.

ADDITIONAL EXPERIMENT

Staurosporine Induces Apoptosis and p34^(cdc2) Kinase

Staurosporine has been reported to cause the rapid death by apoptosis ofa number of cell types (e.g. Jacobson et al., Nature 361 365 (1993);Falcieri et al., Biochem. Biophys. Res. Commun. 193 19 (1993); Bertrandet al., Exp. Cell Res. 207 388 (1993)). It now appears that thiscompound induces a dose-dependent decrease in survival of differentiatedPC12 cells in the presence of NGF (FIG. 15). When nuclear morphology wasanalysed by propidium iodide staining, extensive chromatin condensationwas observed, indicating that death in these cells is occurring by theprocess of apoptosis.

Since activation of the cell cycle regulated kinase p34^(cdc2)accompanies the induction of death due to NGF withdrawal in these cells,the question of whether staurosporine-induced death of PC12 cells alsoresults in the activation of this enzyme was investigated. Addition ofstaurosporine (1 μM) to differentiated PC12 cells activated p34^(cdc2)within 5 min, which continued to increase over the 4 h time period. Thefirst evidence of pyknotic nuclei was not observed until 2 h afterstaurosporine addition, and therefore indicates that activation of thiskinase clearly precedes chromatin condensation (FIG. 16).

It has been shown that staurosporine causes death of fibroblasts andthat overexpression of the protein bcl-2 can substantially delay thiseffect (Jacobson et al Nature 361, 365 (1993)). Staurosporine treatmentof either proliferating (FIG. 17), or serum-deprived rat1 (FIGS. 18A and18B) or Swiss 3T3 (FIG. 19) fibroblasts also causes activation ofp34^(cdc2), which is significantly reduced if these cells are forced tooverexpress bcl-2 (FIGS. 17, 18A & B and 19). This could suggest thatbcl-2 lies upstream of p34^(cdc2) in the death pathway, and indicate apotential mechanism for the action of cbl-2.

Overall, these results demonstrate that activation of p34^(cdc2) isoften a part of the apoptotic pathway in both serum-deprived andproliferating cells. These data are reinforced by the recent observationthat both cytotoxic T-lymphocyte- and staurosporine-induced death oftarget cells also involves activation of this kinase (Shi et al.,Science 263 1143 (1994). Since staurosporine is a general kinaseinhibitor, the ability of this compound to induce both death andactivation of the kinase p34^(cdc2) is especially important.

What is claimed is:
 1. A method of treating or preventing aneurodegenerative disease involving apoptotic cell death, the methodcomprising administering to a subject in need thereof, or to cells of asubject, an effective amount of an agent which prevents cell entry into,or passage through, the mitotic cycle, wherein the agent prevents orinhibits cyclin synthesis as identified by a reporter gene assay usingthe cyclin promoter, or an enzymatic assay for cdc2 or cdk2 activationor cdc2 or cdk2 activity, and wherein the cell death occurs innon-dividing cells; provided that the agent is not bFGF, IGF-I, IGF-II,potassium ions or a cAMP-elevating agent.
 2. A method as claimed inclaim 1, wherein the gent prevents or inhibits the activation ofp34^(cdc2) as identified by an enzymatic assay for an activator ofp34^(cdc2).
 3. A method as claimed in claim 1, wherein the agent is apromoter or activator of wee1 or mik1 as identified by an enzymaticassay for an activator of wee1 or mik1.
 4. A method as claimed in claim1, wherein the agent is an inhibitor of cdc25 as identified by anenzymatic assay for cdc25 activity.
 5. A method as claimed in claim 1,wherein the agent is an inhibitor of cyclin B as identified by anenzymatic assay for cyclin B activity.
 6. A method as claimed in claim1, wherein the agent is serotonin, dopamine, ascorbic acid, gliquidone,caffeine, hydrocortisone or dexamethasone.
 7. A method as claimed inclaim 1, wherein the non-driving cells are neurons.
 8. A method asclaimed in claim 7, which is a method for the treatment or prophylaxisof Alzheimer's disease, Parkinson's disease, motor-neuron disease, orneuronal cell death in stroke.
 9. A method as claimed in claim 1,wherein the agent is administered orally.
 10. A method of treating orpreventing a neurodegenerative disease involving apoptotic cell death,the method comprising administering to a subject in need thereof, or tocells of a subject, an effective amount of 6-dimethylaminopurine(6-DMAP) or an analogue thereof which prevents cell entry into, orpassage through, the mitotic cycle and wherein the cell death occurs innon-dividing cells.
 11. A method of preventing apoptotic cell death, themethod comprising administering to cells an effective amount of anagent, other than bFGF, IGF-I, IGF-II, potassium ions or acAMP-elevating agent, which prevents cell entry in to the mitotic cycle,wherein the agent prevents or inhibits cyclin synthesis as identified bya reporter gene assay using the cyclin promoter, or an enzymatic assayfor cdc2 or cdk2 activation or cdc2 or cdk2 activity, and wherein thecell death occurs in non-dividing cells.
 12. A method of preventingapoptotic cell death in a research model, the method comprisingadministering to cells an effective amount of an agent which preventscell entry into, or passage through, the mitotic cycle, wherein theagent prevents or inhibits cyclin synthesis as identified by a reportergene assay using the cyclin promoter, or an enzymatic assay for cdc2 orcdk2 activation or cdc2 or cdk2 activity, and wherein the cell deathoccurs in non-dividing cells; provided that the agent is not bFGF, IGF-Ior IGF-II, potassium ions or a cAMP-elevating agent.
 13. The method asclaimed in claim 12, wherein the research model is on the etiology ofcell death.
 14. The method as claimed in claim 12, wherein the methodfurther comprises using the agent to prevent or inhibit cyclin synthesisor cdc2 or cdk2 activation or cdc2 or cdk2 activity.
 15. A method asclaimed in claim 12, wherein the agent prevents or inhibits theactivation of p34^(cdc2).
 16. A method as claimed in claim 12, whereinthe agent is 6-dimethylaminopurine (6-DMAP) or an analogue thereof. 17.A method as claimed in claim 12, wherein the agent is a promoter oractivator of wee1 or mik1.
 18. A method as claimed in claim 12, whereinthe agent is an inhibitor of cdc25.
 19. A method as claimed in claim 12,wherein the agent is an inhibitor of cyclin B.
 20. A method as claimedin claim 12, wherein the agent is serotonin, dopamine, ascorbic acid,gluquidone, caffeine, hydrocortisone or dexamethasone.
 21. A method asclaimed in claim 12, wherein the non-dividing cells are muscle cells.22. A method as claimed in claim 12, wherein the non-dividing cells areneurons.